Uncontrolled cell division is at the heart of the cancer problem and inaccurate chromosomal segregation is causal in several congenital malformations. It is possible that an improved understanding of mitosis will lead to new approaches in cancer chemotherapy and to control of abnormal chromosomal segregation. This proposal concerns mitotic spindle function in lysed mammalian tissue culture cells. Cells lysed early in anaphase into a mixture of polymerizable microtubule protein will continue to move chromosomes towards the spindle poles. This system will be used to characterize spindle biochemistry and to identify the enzymes responsible for chromosome movement. The steps required to reach this goal are: 1) development of a lysis protocol that maintains chromosome motion at consistent rates from one experiment to the next. Measurements of spindle mass, spindle birefringence and cytoplasmic density will be used as criteria for extent of lysis and extraction; 2) the enzymes responsible for chromosome movement will be identified by adding jamming proteins (proteins that bind to actin, myosin, dynein, etc.) or contractile proteins to perturb spindle function. The physiological requirements (ion cofactor, nucleotides, etc.) of chromosome movement will be established; 3) determination of actin filament and microtubule polarity in the spindle. Heavy meromyosin will be added directly to lysed cells to determine actin filament polarity. I will attempt to develop a polarity probe for microtubules using short microtubule fragments grown off decorated seeds as markers. This information should contribute significantly to our understanding of how mitosis works at a molecular level.